All vitamin A that is present in higher animals must be derived from the diet. Provitamin A carotenoids like beta-carotene, act essentially in man and other species as precursors for vitamin A synthesis. In addition, dietary carotenoids may have other beneficial actions that are independent of their provitamin A activity. Although carotenoid uptake and carotenoid actions have been systematically studied for over 40 years, very little detailed biochemical information regarding the uptake and metabolism of these substance is presently available. This lack of biochemical information has in large part been due to technical difficulties in purifying and cloning proteins that are importantly involved in carotenoid uptake and metabolism. In the last year, we reported the cloning and characterization of a mudne cDNA that encodes an enzyme that when expressed in either E. coli or CHO cells confers the ability to cleave beta-carotene to vitamin A aldehyde. We now propose to investigate the biochemistry and physiology of mouse and human carotene cleavage enzyme (CCE) and to explore the uptake and metabolism of carotenoids in mutant mice where levels of this enzyme are manipulated genetically. Our proposal consists of 4 Specific Aims. In Aim 1, we will identify proteins present in tissues that express CCE that interact physiologically with this enzyme. Here we are interested in understanding how a soluble enzyme like CCE obtains and cleaves extremely insoluble carotenoids to insoluble retinoid products. Based on our data, we hypothesize that other as yet unidentified cellular protein(s) must play a role in metabolically channeling carotene cleavage towards vitamin A formation. Aim 2 proposes a systematic study of the tissue and cellular distribution of CCE protein and mRNA levels in the adult, post-natal and embryonic mouse. In addition, we will investigate how nutritional and hormonal factors influence enzyme expression in the mouse. In Aim 3, we propose to generate and study tissue-specific knockout mice for CCE. These studies will allow us to identify and characterize the essential and nonessential actions of this enzyme in living organisms. In Aim 4, we propose to use a cDNA clone for human CCE to characterize the biochemical properties of the recombinant enzyme.